Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized inclusion structures concentrate viral replication proteins and nucleic acids, prevent activation of cell-intrinsic defenses, and coordinate release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in knowledge about how these organelles form and mediate their functions. The proposed research uses reovirus, a genetically tractable experimental model that shows promise for oncolytic and vaccine applications, to elucidate mechanisms of double-stranded (ds) RNA virus inclusion formation, genome replication, and progeny particle release. Like other dsRNA viruses, which include important pathogens of animals (orbiviruses) and humans (rotaviruses), reovirus inclusions are nucleated by viral nonstructural proteins that recruit viral structural proteins for genome replication and particle assembly. We have discovered that reovirus inclusions are embedded in lipid and that progeny reovirus particles are transported from inclusions and released from some types of cells using a mechanism that does not cause cell lysis. Three integrated specific aims are proposed to fill key knowledge gaps about reovirus inclusion biogenesis and function. In Specific Aim 1, mechanisms underlying formation of reovirus inclusions will be determined using correlative light and electron microscopy coupled with electron tomography and biochemical analyses. Membrane-biosynthetic pathways required for reovirus inclusion formation will be identified using pharmacologic inhibitors and gene deletions of candidate host molecules. In Specific Aim 2, functions of reovirus nonstructural protein ?NS, an essential reovirus inclusion component that binds viral RNA and GTP, will be defined using in vitro assays of RNA binding and kinase activity. The structure of ?NS alone and in complex with its ligands will be determined using X-ray crystallography and cryo-electron microscopy. Activities of ?NS in reovirus replication will be determined using viruses with structure-guided mutations in ?NS engineered by reverse genetics and inhibitors of ?NS GTP-binding activity. In Specific Aim 3, pathways used by reovirus to exit infected cells will be elucidated using electron tomography and target-specific molecular probes. Candidate vesicular pathways will be tested for function in reovirus egress using RNAi, pharmacologic inhibitors, and dominant-negative mutants. New host mediators of reovirus exit will be identified using an RNAi-based viral egress screen. These studies will enhance a basic understanding of mechanisms by which pathogenic viruses alter cellular architecture to engineer inclusion organelles, replicate their genomes, and exit infected cells. We anticipate that this information will foster development of antiviral drugs that impede these essential viral replication steps and enhance the use of reovirus as an oncolytic therapeutic.